UMD Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/3
New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a given thesis/dissertation in DRUM.
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
5 results
Search Results
Item Integration and Competition in Immune Cell Models(2022) Bull, Abby L; Losert, Wolfgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cell motility plays an integral role in most biological processes. One principle of motility is the protrusions and retractions of cellular membranes called pseudopods. The physical force behind pseudopod formation is actin polymerization. As the physical driver, actin polymerization integrates the cells’ upstream biochemical signaling cascades and turns the signals into action as the combined output and readout of the state of the cell. Actin polymerization not only occurs within pseudopods but propagates throughout a cell in waves that can be understood and modeled as excitable media.This dissertation focuses on actin wave dynamics in the context of directed cell mi- gration using both experimental and numerical techniques. The directional guidance of cell migration is essential in physiological processes including embryonic development, cancer metastasis, and wound healing. In this thesis, I analyze how immune-like cells are guided by external stimuli that are common in wound environments: electric fields, chemical gradients, and surface texture. A focus of this work is on the emergent excitable wave behavior of actin, and the pseudopods they generate, in simplified, in vitro environments. Actin waves have been previously shown to respond to and be guided by the topography of an underlying substrate. Additionally, static quantifications of actin filaments during or after electric field stimulation have shown that filaments become asymmetrically distributed within the cytoplasm. In neutrophils, the combination of cues leads to higher control of cell motility by guiding the internal actin waves. Using an optical flow algorithm, I quantify the actin waves on multiple length scales to ascertain the role of each guidance cue in affecting cell motion. I find that the waves preferentially polymerize near and travel along the nanoridges. Actin waves nucleate preferentially on the cathode side and reorient the cell’s axis of polarity (i.e., the position of the dominant pseudopod). The second example of competing guidance cues involves studying the collective motion of cells in response to cell-cell signal relay in competition with surface topography. I use Dictyostelium discoideum cells as a model system for this work, as they migrate collectively due to signal relay. The signal relay of these cells is similar to many immune cell species. Using a combination of image analysis tools and a coarse-grained stochastic model, I find that guidance by nanoridges overrides the chemical signal relay and forces cells to migrate individually, suppressing streaming behavior. I model both the secretion and propagation of chemical signals using an excitable systems framework. This work highlights that bidirectional signals can be effective at suppressing cell-cell attraction and streaming motion. The response of immune cells to external stimuli in the wound environment is not universal. Macrophages, one of the largest immune cells, are observed to migrate away from the wound upon wound-induced electric field generation. In the third example, I study actin dynamics of M0 (resting) macrophage cells to elucidate how these cells interact with external electric fields. This cell type exhibits oscillatory actin waves at rest. With electric field stimulation, the oscillatory actin waves start to generate protrusions. Often, the protrusions begin with actin-depleted regions, indicating that contractile ele- ments are involved in conjunction with overall cell volume conservation. This thesis highlights the different methods in which actin waves integrate external cues, specifically electric fields, into cell responses that are cell-type specific.Item THE ENDOGENOUS REGULATION OF THE HUMAN MACROPHAGE ACTIVATION RESPONSE(2020) Hamidzadeh, Kajal; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Macrophages are innate immune cells that participate in host defense to invading pathogens. They are powerful producers of cytokines and inflammatory mediators due to their efficient recognition of pathogen associated molecular patterns (PAMPs) via toll like receptors (TLRs). We and others have shown that the activation response to PAMPs is transient. In the present work, we demonstrate that stimulated macrophages produce adenosine and prostaglandin E2, which function as regulators of the macrophage activation response. Macrophages also upregulate receptors for these regulators to terminate inflammation and promote wound healing. We performed high throughput RNA sequencing to characterize the transcriptomes of human monocyte-derived macrophages in response to stimulation with LPS + Adenosine or LPS + PGE2. These cells exhibited a decrease in inflammatory transcripts and an increase in transcripts associated with cell growth and repair when compared to cells stimulated in the absence of these regulators. Macrophages can be generated from precursor cells in response to two different growth factors; M-CSF (macrophage colony stimulating factor) and GM-CSF (granulocyte-macrophage colony stimulating factor). M-CSF is expressed constitutively in a variety of tissues, while GM-CSF is expressed primarily in the lung, but can be induced in other tissues under inflammatory conditions. We demonstrate that human macrophages differentiated in M-CSF readily adopt an anti-inflammatory, growth promoting phenotype in response to LPS + Adenosine or LPS + PGE2, while macrophages differentiated in GM-CSF do not. This observation suggests that M-CSF derived human macrophages may be better able to alter their activation state in response to surrounding signals in order to maintain homeostasis. GM-CSF derived macrophages, in contrast, may undergo a more prominent activation response that is associated with inflammation and tissue destruction due to their inability to efficiently respond to resolving molecules.Item Transcriptomic profiling of Leishmania parasites and host macrophages during an infection(2015) Dillon, Laura Anne Liefer; El-Sayed, Najib M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Leishmania parasites cause leishmaniasis, a group of diseases that range in manifestations from skin lesions to fatal visceral disease. The parasite's life cycle is divided between its insect vector and its mammalian host, where it resides primarily inside of macrophages. Once intracellular, Leishmania parasites must avoid being killed by the innate and adaptive immune responses. We performed transcriptomic profiling using RNA-seq to simultaneously identify global changes in gene expression in Leishmania parasites across multiple lifecycle stages and in infected macrophages from both murine and human hosts. Using a novel approach based on a dual statistical test to identify genes that were differentially expressed relative to both uninfected macrophages and macrophages that had ingested inert particles, we were able to filter out genes that were differentially regulated as part of a general phagocytic response and thereby select genes specific to Leishmania infection. The most substantial and dynamic Leishmania-specific differential expression responses were observed during early infection, while changes observed later were common to phagocytosis more generally. An evaluation of RNA processing events within the parasite revealed precise UTR boundaries for a majority of genes and widespread alternative trans-splicing and polyadenylation. Collection of data from multiple biological replicates, the use of matched host control samples, careful statistical analysis of variation, and removal of batch effects enabled the detection of biological differences between samples and timepoints with high confidence and sensitivity. Pathway and gene ontology analyses provided insights into the higher level processes activated across parasite developmental stages and during intracellular infection to reveal signatures of Leishmania differentiation and infection.Item THE IDENTIFICATION AND CHARACTERIZATION OF AN INTRINSIC CD39/A2R-BASED REGULATORY MECHANISM THAT GOVERNS MACROPHAGE ACTIVATION RESPONSES(2014) Cohen, Heather Bloom; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Macrophages are acutely sensitive to changes within their environment and can readily develop into a variety of activation states important for both the progression and resolution of inflammation. In response to immunological threats, macrophages must be able to effectively clear infections without sacrificing the integrity of the affected tissue. Thus, these cells must successfully balance their activation responses in order to preserve tissue function and the overall health of the host. The failure to properly regulate macrophage activation responses often manifests in the clinic in a variety of disease scenarios including sepsis, chronic inflammatory disorders, and cutaneous Leishmaniasis. While many factors that drive the initiation of macrophage activation are known, it remains unclear what governs the transition to an immunosuppressive state. This study reveals that macrophages can control their own activation status through the coordination between the ecto-ATPase, CD39, and the adenosine 2a and 2b receptors (A2Rs). The first part of this work shows that soon after toll-like receptor (TLR) stimulation, macrophages secrete and convert ATP into immunosuppressive adenosine via CD39. Moreover, we show that CD39 on macrophages is necessary to induce regulatory macrophage development and prevent severe immunopathology in a mouse model of septic shock. The next sets of data demonstrate that TLR activation also enhances A2bR expression, thus completing the CD39-initiated autoregulatory circuit to limit inflammatory macrophage responses. The second part of this work demonstrates that the chronic inflammatory disease-asociated cytokine, IFN-gamma, prevents TLR-induced A2bR expression and consequently promotes the hyper-production of inflammatory cytokines by macrophages thereby revealing a novel mechanism by which IFN-gamma; maintains overactive macrophages. The final chapter illustrates that while the A2bR is the dominant adenosine receptor mediating the inhibition of inflammatory cytokine production, A2aR signaling inhibits nitric oxide generation and that its expression may be hijacked by intracellular parasites to evade innate host defense mechanisms. Thus, this study demonstrates that inflammatory macrophage activation is inherently transient and that macrophages can reprogram themselves. These results culminate in the discovery of a novel immunomodulatory mechanism reliant on macrophage purinergic signaling and offer new targets and strategies to more effectively treat myriad inflammatory and infectious diseases.Item Defining Critical Parameters for Producing and Modulating Inflammation Caused by Cell Encapsulating Alginate Microspheres(2007-09-11) Breger, Joyce Catherine; Wang, Nam Sun; Lyle, Dan B; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Minimizing induced inflammation, particularly nitric oxide (NO) production, is critical to optimal function or failure of implanted encapsulated cells. The purpose of this study is to define critical factors that affect toxic NO production from the macrophage cell line RAW264.7 in response to alginate microcapsules. The effects of the following were determined: 1) concentration of endotoxin (LPS) contamination; 2) presence of interferon-gamma (IFN-γ); 3) bead diameter and alginate volume; and 4) anti-inflammatory drugs in the alginate. A higher concentration (5 X) of LPS was required in alginate to produce the effect seen by LPS free in medium, sensitivity was enhanced by IFN-γ, bead diameter was inversely proportional to NO2 under low inflammatory conditions, and parthenolide in alginate significantly reduced inflammation. These results suggest that survival of implanted encapsulated cells may be improved by using highly purified alginate, avoiding ancillary inflammation, controlling surface area presentation, and incorporating anti-inflammatory drugs into the capsule matrix.